Molecular mechanisms of chaperone‐directed protein folding: Insights from atomistic simulations

Abstract Molecular chaperones, a family of proteins of which Hsp90 and Hsp70 are integral members, form an essential machinery to maintain healthy proteomes by controlling the folding and activation of a plethora of substrate client proteins. This is achieved through cycles in which Hsp90 and Hsp70, regulated by task‐specific co‐chaperones, process ATP and become part of a complex network that undergoes extensive compositional and conformational variations. Despite impressive advances in structural knowledge, the mechanisms that regulate the dynamics of functional assemblies, their response to nucleotides, and their relevance for client remodeling are still elusive. Here, we focus on the glucocorticoid receptor (GR):Hsp90:Hsp70:co‐chaperone Hop client‐loading and the GR:Hsp90:co‐chaperone p23 client‐maturation complexes, key assemblies in the folding cycle of glucocorticoid receptor (GR), a client strictly dependent upon Hsp90/Hsp70 for activity. Using a combination of molecular dynamics simulation approaches, we unveil with unprecedented detail the mechanisms that underpin function in these chaperone machineries. Specifically, we dissect the processes by which the nucleotide‐encoded message is relayed to the client and how the distinct partners of the assemblies cooperate to (pre)organize partially folded GR during Loading and Maturation. We show how different ligand states determine distinct dynamic profiles for the functional interfaces defining the interactions in the complexes and modulate their overall flexibility to facilitate progress along the chaperone cycle. Finally, we also show that the GR regions engaged by the chaperone machinery display peculiar energetic signatures in the folded state, which enhance the probability of partial unfolding fluctuations. From these results, we propose a model where a dynamic cross‐talk emerges between the chaperone dynamics states and remodeling of client‐interacting regions. This factor, coupled to the highly dynamic nature of the assemblies and the conformational heterogeneity of their interactions, provides the basis for regulating the functions of distinct assemblies during the chaperoning cycle.

Here, lighter pixels correspond to highly coordinated residue pairs, while darker ones report on low coordina3on pairs.(C) DF difference matrix between the Loading-ADP matrix and the Loading-APO matrix.The axes report the residue numbers and the protein/domain division.In the difference matrix red pixels represent a more rigid coordina3on of the Loading-ADP pair residue respect to the Loading-APO pair residue, while the black pixels represent a less coordina3on of the Loading-ADP pair residue respect to the Loading-APO pair residue.The white pixels represent the same coordina3on, instead.Coloured boxes in panel C refer to Figure 3 (C-E) in main text.Average C devia3on (Å) for each residues between the equilibrium Loading-ADP simula3ons and the non-equilibrium APO simula3ons.The average devia3on is obtained from the 176 non-equilibrium simula3ons windows at 5 ns aVer ADP removal (see Method for details).On the x-axis are reported the single components of the mul3protein complex (the numera3on corresponds to the one used in our simula3ons).

Loading-ADP
Loading-APO 180°1 80°F igure S3.In the leV por3on of the picture there are the RMSF plots of the whole trajectories.In detail, the upper plot refers to Loading-ADP system, while the lower refers to the Loading-APO.The right por3on of the picture contains the 3D structures of the loading complex, on which RMSF values are projected.Higher is the color intensity, higher is the RMSF value.The upper representa3ons refer to the front and rear view of the loading complex in the Loading-ADP simula3ons, while the lower respresenta3ons refer to the same views of the loading complex, but in the Loading-APO simula3ons, instead.The difference matrix has a color bar that goes from red to black.Red colored pixels represent a more rigid coordina3on of the Loading-ADP pair residue respect to the Loading-Y354E pair residue, while the black pixels represent a less coordina3on of the Loading-ADP pair residue respect to the Loading-Y354E pair residue.The white pixels represent the same coordina3on rigidity, instead.  1 for simula3on labels).Here, lighter pixels correspond to highly coordinated residue pairs, while darker ones report on low coordina3on pairs.On the axes there are the residue numbers (sequence) and the protein/domain division.

RMSD (Å)
RMSD (Å)    1 for simula3on labels).The axes report residue numbers.In the lower panel are reported the 3-D structures of GR in the two different systems.The green box in the matrices iden3fies the GR's tail regions that gain coordina3on in terms of DF values.

GR-maturation GR-alone
Stabilization High Low

60°
Table S1.List of residues that define the binding pockets iden3fied for already known allosteric ligands for Hsp70 and Hsp90.

Figure S1 .
Figure S1.Full-detail Residue-Pair Distance fluctua3ons matrix of the (A) Loading-ADP system and of the (B) Loading-APO system.Here, lighter pixels correspond to highly coordinated residue pairs, while darker ones report on low coordina3on pairs.(C) DF difference matrix between the Loading-ADP matrix and the Loading-APO matrix.The axes report the residue numbers and the protein/domain division.In the difference matrix red pixels represent a more rigid coordina3on of the Loading-ADP pair residue respect to the Loading-APO pair residue, while the black pixels represent a less coordina3on of the Loading-ADP pair residue respect to the Loading-APO pair residue.The white pixels represent the same coordina3on, instead.Coloured boxes in panel C refer to Figure 3 (C-E) in main text.
Figure S2.Average C devia3on (Å)  for each residues between the equilibrium Loading-ADP simula3ons and the non-equilibrium APO simula3ons.The average devia3on is obtained from the 176 non-equilibrium simula3ons windows at 5 ns aVer ADP removal (see Method for details).On the x-axis are reported the single components of the mul3protein complex (the numera3on corresponds to the one used in our simula3ons).

Figure S4 .Figure S5 .Figure S6 .
Figure S4.In the image sequence, from leV to right, it is represented the Hsp70CSBD and GR tail movement that occurs during the simula3ons of the Loading-ADP.

Figure S7 .
Figure S7.Full Distance Fluctua3ons matrix of the Matura3on system (see Table1for simula3on labels).Here, lighter pixels correspond to highly coordinated residue pairs, while darker ones report on low coordina3on pairs.On the axes there are the residue numbers (sequence) and the protein/domain division.

Figure S8 .Figure S9 .
Figure S8.RMSD plots referred to the Matura3on system simula3ons.In detail, the upper plot represent the RMSD calculated on the NTD-A (black) and NTD-B (red) of Hsp90 when the trajectory is aligned on the protein backbone of Hsp90 except for NTD-A.The boaom plot, instead, represent the RMSD calculated on the NTD-A (black) and NTD-B (red) of Hsp90 when the trajectory is aligned on the protein backbone of Hsp90 except for NTD-B.

Figure S12 .
Figure S12.Distance Fluctua3on pairwise matrices for the client GR simula3ons (extracted from Loading-ADP and Matura3on simula3on; see Table1for simula3on labels).The axes report residue numbers.In the lower panel are reported the 3-D structures of GR in the two different systems.The green box in the matrices iden3fies the GR's tail regions that gain coordina3on in terms of DF values.

Figure S13 .
Figure S13.MLCE analysis on different GR state (leV: Loading-ADP; right: Matura3on).Each panel shows the regions that are energe3cally less prone to unfold, the folding core.Backbone thickness of each residue and colour intensity is propor3onal to the calculated energy coupling value (see Methods).